In the field of proteomic analysis and pathological examination, there is emerging a new analytical method for completely visualizing a protein expressed in a tumor tissue and the like on a cellular level.
Mass spectrometry is one of the most effective methods to visualize the inner structure of a cell. In mass spectrometry, laser desorption/ionization (LDI) and matrix assisted laser desorption/ionization (MALDI) methods have been widely used in recent years as an ionization method in the life science field because of its capability of suppressing the cleavage of a high molecular compound with a large molecular weight. According to the above ionization methods, a laser beam irradiates a sample to promote charge movement inside a substance absorbing the laser beam for ionization. The ionized molecules are analyzed by an analyzing unit such as a time-of-flight mass spectrometer to detect the mass/charge (m/Z) ratio. The mass spectrometry using these ionization methods can acquire a component distribution while moving the laser irradiation position on a surface of sample. The obtained fine and two-dimensional mass spectrum is called a mass spectrum image. This imaging is a promising means for obtaining a protein distribution in a cell.
Secondary ion mass spectrometry (SIMS) is an analysis method of obtaining a two-dimensional mass spectrum by ionizing atoms and molecules by irradiating a sample with primary ions. The SIMS is classified into a dynamic-SIMS and a static-SIMS based on the difference in amount of primary ion irradiation. The dynamic-SIMS sputters a sample surface with a large amount of primary ion irradiation to generate a large amount of secondary ions. In contrast to this, the static-SIMS uses the amount of primary ion irradiation sufficiently smaller than the number of molecules composing the surface to generate secondary ions maintaining the molecular structure. This method is more advantageous in analyzing organic compounds than the dynamic-SIMS since abundant information about the molecular structure can be obtained. The SIMS can also form a mass spectrum image by performing mass spectrometry while moving the position of primary ion irradiation.
The present inventors have proposed various methods based on high spatial resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS method) which is a kind of static-SIMS, such as a method of decomposing the surface layer of a body tissue section by digestive enzyme and measuring the two-dimensional distribution of the generated peptide fragments and a method of detecting a minute amount of body-related substance using a SIMS-specific sensitizer.
Note that a present mass spectrum image is obtained by integrating a desired signal intensity vs mass/charge from a mass spectrum obtained at each position on the sample and simply two-dimensionally displaying the integrated signal intensity at each position.
In general, an image acquired by an apparatus is a blurred one depending on the instrument for observation and the object to be observed. An image having a blur is called a blurred image or a deteriorated image. For example, in an optical system having a lens such as a microscope, a telescope or a digital camera, image blurring is caused by distortion, lens aberration, and an out-of-focus state.
In order to reduce image blurring, an image restoration algorithm is generally used. More specifically, the cause of image blurring is treated as a function and numerical processing is performed thereon to reduce image blurring. Here, a function defining the cause of image blurring is referred to as “blurring function”. For example, in the above described image restoration method for blurred images in a light microscope, image restoration is generally performed by using a preliminarily measured point spread function of lens as a blurring function.
Japanese Patent Application Laid-Open No. 2008-177064 (PTL1) discloses a method of performing restoration of an image obtained by a scanning electron microscope and a scanning ion microscope by calculating a blurring function according to the imaging condition such as an accelerating voltage, a beam aperture angle, a beam current, or a focus position. Further, an effect of charged-particle beam diffusion in the sample on image blurring is considered.
The mass spectrometry imaging also generates image blurring. However, the image blurring is generated not only for the same reason as a general microscope, but also by a the cause specific to the mass spectrometry. More specifically, the SIMS uses sputtering phenomena and thermal desorption phenomena. Therefore, image blurring occurs by a cause depending on primary ions, such as ionic species and energy, as well as by a cause depending on atoms and molecules to be observed.
When a macromolecule such as a protein is ionized by primary ion irradiation, the molecules are ionized with their molecular structure intact or fragmented. Whether fragmentation occurs or not, depends on the distance from the position of primary ion incidence. The detailed description of this phenomenon is found in “D. Rading, et al, J. Vac. Sci. Technol., A vol. 18, p. 312 (2000)” (NPL1).